Bone stimulating factor

ABSTRACT

Polypeptides which stimulate bone growth: Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Pro (SEQ ID NO:1) and subsequences, particularly Arg Thr Asn Glu His Thr Ala Asp Cys Lys (SEQ ID NO:9) and associated nucleotide sequences, methods of preparation and use, antibodies and kits.

This application is a continuation-in-part of prior application Ser. No.08/487,074, U.S. Pat. No. 5,880,094, filed Jun. 7, 1995 which isincorporated herein by reference.

The present invention relates to polypeptides which stimulate bonegrowth.

Understanding of issues related to bone growth and strength hasprogressed over the years, a summary being provided in internationalpatent application No. PCT/CA 94/00144, published under internationalpublication No. WO 94/20615 on Sep. 15, 1994.

Various approaches to treatment of diseases involving reduction of bonemass and accompanying disorders are exemplified in the patentliterature. For example, U.S. Pat. No. 4,877,864, issued Oct. 31, 1989describes human and bovine "bone inductive factors." Internationalpatent application published Sep. 17, 1992 under Ser. No. 92/15615describes a protein derived from a porcine pancreas which acts todepress serum calcium levels for treatment of bone disorders that causeelevation of serum calcium levels. European Patent Application No. 504938 published Sep. 23, 1992 describes the use of di- or tripeptideswhich inhibit cysteine protease in the treatment of bone diseases.International patent application published Sep. 3, 1992 under Ser. No.92/14481 discloses a composition for inducing bone growth, thecomposition containing activin and bone morphogenic protein. EuropeanPatent Application No. 499 242 published Aug. 19, 1992 describes the useof cell growth factor compositions thought to be useful in bone diseasesinvolving bone mass reduction because they cause osteoblastproliferation. International patent application published Jun. 25, 1992under Ser. No. 92/10515 1992 describes a drug containing the humanN-terminal parathyroid hormone (PTH) fragment 1-37. European PatentApplication No. 451 867 published Sep. 16, 1991 describes parathyroidhormone peptide antagonists for treating dysbolism associated withcalcium or phosphoric acid, such as osteoporosis. U.S. Pat. No.5,461,034 issued Oct. 24, 1995 to Yissum Research Development Company ofthe Hebrew University of Jerusalem describes osteogenic growthpolypeptides identified from regenerating bone marrow.

A relatively short half life of PTH in the blood serum and the positiveeffect of intermittent PTH injection on bone volume led the presentinvestigator to the hypothesis that PTH may in some way lead toinduction of a second factor into the circulatory system. The presenceof such a second factor in blood serum of rats and of humans has thusbeen investigated.

It has been found possible to isolate from rat blood serum a polypeptidesubstance which, upon administration to rats incapable of producing PTH(parathyroidectomized rats), produces an increase in the observed bonemineral apposition rate. A nucleic acid probe, based on the amino acidsequence of the rat peptide was synthesized and used to screen a humanliver cDNA fetal library in order to isolate a human nucleic acidsequence coding for a human bone apposition polypeptide. A polypeptidederived from the nucleic acid sequence was thus chemically synthesizedaccording to the derived sequence Gly Ile Gly Lys Arg Thr Asn Glu HisThr Ala Asp Cys Lys lie Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu ThrLeu Met Val Leu Asp Gln Asn Gin Pro (SEQ ID NO:1). It has been observedthat the bone apposition rate in intact rats increases in a dosedependent fashion upon administration of this chemically synthesizedcompound. Reduced bone growth, normally observed for ovariectomizedrats, was observed not to occur in rats after being administered withthe polypeptide over a four week period beginning two weeks afterovariectomization. Bone calcium density was found to be maintained inovariectomized rats administered with the polypeptide over an eight weekperiod beginning eight weeks after ovariectomization.

It is thought possible that the active polypeptide is a dimer of theforegoing sequence, there being evidence of significant dimer formation,presumably due to a disulfide bridge between two polypeptides having thesequence shown.

A modified form of the polypeptide containing a cys-ala substitution wasthus synthesized: Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp AlaLys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val LeuAsp Gin Asn Gln Pro (SEQ ID NO:3). Some of the bone stimulatory effectsof the "normal" polypeptide (SEQ ID NO:1) were found for the modifiedpolypeptide.

In other experiments, the bone mineral apposition rate in ratsadministered with rabbit antibodies to the normal polypeptide (SEQ IDNO:1) was found to be suppressed. The suppression was found to beattenuated in rats administered with both the normal polypeptide andantibodies to same.

Further, certain polypeptide fragments of the normal polypeptide (SEQ IDNO:1) have been synthesized and each has been found to have bonestimulatory effects:

SEQ ID NO:4:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val

SEQ ID NO:5:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu His Lys Lys Ala Ala

SEQ ID NO:6:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu

SEQ ID NO:7:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile

SEQ ID NO:8:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys

SEQ ID NO:9:

Arg Thr Asn Glu His Thr Ala Asp Cys Lys

Further, the polypeptide identified as SEQ ID NO: 7 has been found toincrease bone calcium content of ovariectomized rats when administeredover a period of eight or twelve weeks.

Other polypeptide fragments of the normal polypeptide (SEQ ID NO: 1)have also been synthesized and have been found to lack the bonestimulatory effect found for the normal polypeptide:

SEQ ID NO:10:

Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln

SEQ ID NO:11:

Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn

SEQ ID NO:12:

Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln

SEQ ID NO:13:

Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp

SEQ ID NO:14:

Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu ThrLeu Met Val Leu Asp

SEQ ID NO:15:

Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His LysLys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn

SEQ ID NO:16:

Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile

The polypeptide identified as SEQ ID NO:9 was modified to include aprotecting group at each end. The N-terminus was thus acetylated andC-terminus was amidated. The activity of this protected polypeptide,identified as SEQ ID NO:24, was found to increase the bone mineralapposition rate in rats beyond that observed for each of thepolypeptides identified as SEQ ID NOS: 1,7 and 9.

It has been reported that histidine and cysteine residues can effectdegradation of asparaginyl- and aspartyl-containing polypeptides in theabsence of catalytic enzymes [Int. J. Peptide Protein Res. 45, 1995,547,553]. The following analogues of the polypeptide identified as SEQID NO:9 were tested, for stability and for effects on bone mineralapposition rate:

SEQ ID NO:25:

CH₃ CO--Arg Thr Asn Glu His Thr Ala Glu Cys Lys--NH₂

SEQ ID NO:26:

CH₃ CO--Arg Thr Gln Glu His Thr Ala Glu Cys Lys--NH₂

SEQ ID NO:27:

CH₃ CO--Arg Thr Gln Glu His Thr Ala Asp Cys Lys--NH₂

In terms of stability under the various conditions tested, thepolypeptides identified as SEQ ID NOS:25 and 26 were found to be morestable than those identified as SEQ ID NOS:9, 7 and 24. The polypeptideidentified as SEQ ID NO:27 was found to be less stable than any of SEQID NOS: 7, 9, 24, 25 and 26.

Each of the polypeptides identified as SEQ ID NOS:24, 25, 26 and 27 werefound to increase the bone apposition rate over that observed forcontrol rats.

Polypeptide sequences corresponding to SEQ ID NOS:25, 26 and 27 in whichthe terminal amino acid residues are not protected are referred toherein as SEQ ID NOS:28, 29 and 30, respectively.

The present invention thus includes a polypeptide having an amino acidsequence corresponding to SEQ ID NO:1 with (a) from one to about four 4amino acids deleted from the N-terminus of SEQ ID NO:1 (b) one to about22 amino acids deleted from the C-terminus of SEQ ID NO:1, or both (a)and (b); or a functionally equivalent homologue. Correspondingly, theinvention includes a polypeptide having an amino acid sequencecorresponding to SEQ ID NO:3 with (a) from one to about four 4 aminoacids deleted from the N-terminus of SEQ ID NO:3 (b) one to about 22amino acids deleted from the C-terminus of SEQ ID NO:3, or both (a) and(b); or a functionally equivalent homologue. Sequence homology inpolypeptides and proteins is understood to those skilled in the art, asdiscussed, for example in Molecular Cell Biology (H. Lodish, D.Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira and J. Darnell,Scientific American Books, New York City, Third Edition, 1995).Likewise, the invention includes a polypeptide having an amino acidsequence corresponding to SEQ ID NO:4 with (a) up to about four 4 aminoacids deleted from the N-terminus of SEQ ID NO:4, (b) up to about 16amino acids deleted from the C-terminus of SEQ ID NO:4, or both (a) and(b); or a functionally equivalent homologue. The invention includes apolypeptide having an amino acid sequence corresponding to SEQ ID NO:5with (a) up to about four 4 amino acids deleted from the N-terminus ofSEQ ID NO:5, (b) up to about 11 amino acids deleted from the C-terminusof SEQ ID NO:5, or both (a) and (b); or a functionally equivalenthomologue. The invention includes a polypeptide having an amino acidsequence corresponding to SEQ ID NO:6 with (a) up to about four 4 aminoacids deleted from the N-terminus of SEQ ID NO:6, (b) up to about 5amino acids deleted from the C-terminus of SEQ ID NO:6, or both (a) and(b); or a functionally equivalent homologue. The invention includes apolypeptide having an amino acid sequence corresponding to SEQ ID NO:7with (a) up to about four 4 amino acids deleted from the N-terminus ofSEQ ID NO:7, (b) up to about 1 amino acids deleted from the C-terminusof SEQ ID NO:4, or both (a) and (b); or a functionally equivalenthomologue. The invention also includes a polypeptide having an aminoacid sequence corresponding to SEQ ID NO:8 with up to about four 4 aminoacids deleted from the N-terminus or a functionally equivalenthomologue. The invention includes a polypeptide having an amino acidsequence corresponding to SEQ ID NO:9 or a functionally equivalenthomologue thereof.

The invention includes a polypeptide up to about 30 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:9 ora functionally equivalent homologue thereof which promotes bone growthin mammals The polypeptide can have a protected terminal amino group, ora protected terminal carboxyl group, or both. The N-terminal protectinggroup can be an acetyl group. The C-terminal can be protected byconversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide of up to about 25 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:9 ora functionally equivalent homologue thereof which promotes bone growthin mammals. The polypeptide can have a protected terminal amino group,or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

Alternatively, the invention includes a polypeptide of up to about 20amino acids in length comprising an amino acid sequence corresponding toSEQ ID NO:9 or a functionally equivalent homologue thereof whichpromotes bone growth in mammals. The polypeptide can have a protectedterminal amino group, or a protected terminal carboxyl group, or both.The N-terminal protecting group can be an acetyl group. The C-terminalcan be protected by conversion of the carboxyl group to an amide group,in which for example, the amino nitrogen thereof is bound to twohydrogen atoms.

The invention includes a polypeptide of up to about 15 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:9 ora functionally equivalent homologue thereof which promotes bone growthin mammals. The polypeptide can have a protected terminal amino group,or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide about 10 amino acids in lengthcomprising an amino acid sequence corresponding to SEQ ID NO:9 or afunctionally equivalent homologue thereof which promotes bone growth inmammals. The polypeptide can have a protected terminal amino group, or aprotected terminal carboxyl group, or both. The N-terminal protectinggroup can be an acetyl group. The C-terminal can be protected byconversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms. Theinvention includes a polypeptide having an amino acid sequencecorresponding to SEQ ID NO:24.

The invention includes a polypeptide up to about 30 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:28or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide of up to about 25 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:28or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

Alternatively, the invention includes a polypeptide of up to about 20amino acids in length comprising an amino acid sequence corresponding toSEQ ID NO:28 or a functionally equivalent homologue thereof whichpromotes bone growth in mammals. The polypeptide can have a protectedterminal amino group, or a protected terminal carboxyl group, or both.The N-terminal protecting group can be an acetyl group. The C-terminalcan be protected by conversion of the carboxyl group to an amide group,in which for example, the amino nitrogen thereof is bound to twohydrogen atoms.

The invention includes a polypeptide of up to about 15 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:28or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide about 10 amino acids in lengthcomprising an amino acid sequence corresponding to SEQ ID NO:28 or afunctionally equivalent homologue thereof which promotes bone growth inmammals. The polypeptide can have a protected terminal amino group, or aprotected terminal carboxyl group, or both. The N-terminal protectinggroup can be an acetyl group. The C-terminal can be protected byconversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms. Theinvention includes a polypeptide having an amino acid sequencecorresponding to SEQ ID NO:25.

The invention includes a polypeptide up to about 30 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:29or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide of up to about 25 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:29or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

Alternatively, the invention includes a polypeptide of up to about 20amino acids in length comprising an amino acid sequence corresponding toSEQ ID NO:29 or a functionally equivalent homologue thereof whichpromotes bone growth in mammals. The polypeptide can have a protectedterminal amino group, or a protected terminal carboxyl group, or both.The N-terminal protecting group can be an acetyl group. The C-terminalcan be protected by conversion of the carboxyl group to an amide group,in which for example, the amino nitrogen thereof is bound to twohydrogen atoms.

The invention includes a polypeptide of up to about 15 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:29or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide about 10 amino acids in lengthcomprising an amino acid sequence corresponding to SEQ ID NO:29 or afunctionally equivalent homologue thereof which promotes bone growth inmammals. The polypeptide can have a protected terminal amino group, or aprotected terminal carboxyl group, or both. The N-terminal protectinggroup can be an acetyl group. The C-terminal can be protected byconversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms. Theinvention includes a polypeptide having an amino acid sequencecorresponding to SEQ ID NO:26.

The invention includes a polypeptide up to about 30 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:30or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide of up to about 25 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:30or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

Alternatively, the invention includes a polypeptide of up to about 20amino acids in length comprising an amino acid sequence corresponding toSEQ ID NO:30 or a functionally equivalent homologue thereof whichpromotes bone growth in mammals. The polypeptide can have a protectedterminal amino group, or a protected terminal carboxyl group, or both.The N-terminal protecting group can be an acetyl group. The C-terminalcan be protected by conversion of the carboxyl group to an amide group,in which for example, the amino nitrogen thereof is bound to twohydrogen atoms.

The invention includes a polypeptide of up to about 15 amino acids inlength comprising an amino acid sequence corresponding to SEQ ID NO:30or a functionally equivalent homologue thereof which promotes bonegrowth in mammals. The polypeptide can have a protected terminal aminogroup, or a protected terminal carboxyl group, or both. The N-terminalprotecting group can be an acetyl group. The C-terminal can be protectedby conversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms.

The invention includes a polypeptide about 10 amino acids in lengthcomprising an amino acid sequence corresponding to SEQ ID NO:30 or afunctionally equivalent homologue thereof which promotes bone growth inmammals. The polypeptide can have a protected terminal amino group, or aprotected terminal carboxyl group, or both. The N-terminal protectinggroup can be an acetyl group. The C-terminal can be protected byconversion of the carboxyl group to an amide group, in which forexample, the amino nitrogen thereof is bound to two hydrogen atoms. Theinvention includes a polypeptide having an amino acid sequencecorresponding to SEQ ID NO:27.

Polypeptides of the present invention can be incorporated into largerpolypeptide sequences in which there is repetition of active sequencesin a single molecule.

The inventive polypeptide can be synthetic and the amino acid sequencecan have a molecular weight in the range of from about 1000 to 4000.

The invention includes a polypeptide having a sequence of amino acidssufficiently duplicative of another, i.e., second polypeptide having anamino acid sequence corresponding to SEQ ID NO:1 (or SEQ ID NO:3) with(a) from one to about four 4 amino acids deleted from the N-terminus ofSEQ ID NO:1 (or SEQ ID NO:3), (b) one to about 22 amino acids deletedfrom the C-terminus of SEQ ID NO:1 or (SEQ ID NO:3), or both (a) and(b), or a functionally equivalent homologue thereof, such that thepolypeptide is encoded by a DNA that hybridizes under stringentconditions with DNA encoding the second polypeptide. The polypeptide canbe up to about 30 amino acids in length, for example, and the sequenceof that polypeptide can be repeated within a larger polypeptide, orcontain other polypeptide sequences which are not by themselvesstimulate bone growth. Such polypeptide could also be up to 25, 20, 15or about 10 amino acids in length.

"Stringent hybridization conditions" takes on its common meaning to aperson skilled in the art here. Appropriate stringency conditions whichpromote nucleic acid hybridization, for example, 6× sodiumchloride/sodium citrate (SSC) at about 45° C. are known to those skilledin the art. The following examples are found in Current Protocols inMolecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6: For 50 mlof a first suitable hybridization solution, mix together 24 mlformamide, 12 ml 20× SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml 100×Denhardt's solution, 2.5 ml deionized H₂ O, 10 ml 50% dextran sulfate,and 0.5 ml 10% SDS. A second suitable hybridization solution can be 1%crystalline BSA (fraction V), 1 mM EDTA, 0.5 M Na₂ HPO₄ pH 7.2, 7% SDS.The salt concentration in the wash step can be selected from a lowstringency of about 2× SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. Both of these wash solutions may contain 0.1% SDS. Inaddition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C. to highstringency conditions, at about 65° C. The cited reference gives moredetail, but appropriate wash stringency depends on degree of homologyand length of probe. If homology is 100%, a high temperature (65° C. to75° C.) may be used. If homology is low, lower wash temperatures must beused. However, if the probe is very short (<100bp), lower temperaturesmust be used even with 100% homology. In general, one starts washing atlow temperatures (37° C. to 40° C.), and raises the temperature by 3-5°C. intervals until background is low enough not to be a major factor inautoradiography.

In another aspect the invention is a synthetic polypeptide having invivo bone stimulatory activity in mammals and which increases mineralcontent (i.e., calcium) in bones of mammals, having an amino acidsequence which is at least about 19% conserved in relation to the aminoacid sequence identified as SEQ ID NO:1 and having at least one aminoacid deleted therefrom, or a functionally equivalent homologue.

The invention includes a synthetic polypeptide having in vivo bonestimulatory activity in mammals and which increases mineral content inbones of mammals, having an amino acid sequence which is at least about22% conserved in relation to the amino acid sequence identified as SEQID NO:1 and having at least one amino acid deleted therefrom.

The invention includes a synthetic polypeptide having in vivo bonestimulatory activity in mammals and which increases mineral content inbones of mammals, having an amino acid sequence which is at least about25% conserved in relation to the amino acid sequence identified as SEQID NO:1 and having at least one amino acid deleted therefrom.

The invention includes a synthetic polypeptide having in vivo bonestimulatory activity in mammals and which increases mineral content inbones of mammals, having an amino acid sequence which is at least about28% conserved in relation to the amino acid sequence identified as SEQID NO:1 and having at least one amino acid deleted therefrom.

The invention includes any of the foregoing synthetic polypeptides inwhich at least six amino acids deleted from the polypeptide sequence; orin which at least eleven amino acids deleted from the sequence; or inwhich at least sixteen amino acids deleted from the sequence; or inwhich at least twenty-one amino acids deleted from the sequence; or inwhich at least twenty-six amino acids deleted from the sequence.

The invention includes a polypeptide having a sequence of amino acidssufficiently duplicative of one of the foregoing synthetic polypeptidessuch that the polypeptide is encoded by a DNA that hybridizes understringent conditions with DNA encoding the synthetic polypeptide.

In another aspect the invention is a polypeptide exhibiting bonestimulatory activity in mammals, the polypeptide having the sequenceidentified as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:24;SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; orSEQ ID NO:30; analogues thereof wherein the amino acids in the sequencemay be substituted, deleted or added, so long as the bone stimulatoryactivity in mammals derived the three dimensional structure of thesequence is preserved; and conjugates of each of the polypeptides oranalogues thereof, wherein if the polypeptide sequence has thatidentified as SEQ ID NO:1, then there is at least one amino acid deletedtherefrom. The invention includes a polypeptide that has a sequence ofamino acids sufficiently duplicative of such a bone stimulatorypolypeptide (or a functionally equivalent homologue thereof) that thepolypeptide is encoded by a DNA that hybridizes under stringentconditions with DNA encoding the bone stimulatory polypeptide.

In another aspect, the invention is a polypeptide that includes an aminoacid sequence that is between 19% and 90% conserved in relation to theamino acid sequence identified as SEQ ID NO:1; or an amino acid sequencethat is between 19% and 86% conserved in relation to the amino acidsequence identified as SEQ ID NO:1; or an amino acid sequence that isbetween 19% and 69% conserved in relation to the amino acid sequenceidentified as SEQ ID NO:1; or an amino acid sequence that is between 19%and 56% conserved in relation to the amino acid sequence identified asSEQ ID NO:1; or an amino acid sequence that is between 19% and 42%conserved in relation to the amino acid sequence identified as SEQ IDNO:1; or an amino acid sequence that is between 19% and 39% conserved inrelation to the amino acid sequence identified as SEQ ID NO:1; or anamino acid sequence that is between 19% and 28% conserved in relation tothe amino acid sequence identified as SEQ ID NO:1; or an amino acidsequence that is between 28% and 90% conserved in relation to the aminoacid sequence identified as SEQ ID NO:1; or an amino acid sequence thatis between 28% and 86% conserved in relation to the amino acid sequenceidentified as SEQ ID NO:1; or an amino acid sequence that is between 28%and 69% conserved in relation to the amino acid sequence identified asSEQ ID NO:1; or an amino acid sequence that is between 28% and 56%conserved in relation to the amino acid sequence identified as SEQ IDNO:1; or an amino acid sequence that is between 28% and 42% conserved inrelation to the amino acid sequence identified as SEQ ID NO:1; or anamino acid sequence that is between 28% and 39% conserved in relation tothe amino acid sequence identified as SEQ ID NO:1; or a functionallyequivalent homologue that has bone stimulatory activity in a mammal.

The polypeptide can be a chimeric bone stimulating factor that includesany of the amino acid sequences described above as part of theinvention.

The invention includes an agent for use in prevention and treatment of abone reduction related disease that includes any polypeptide describedabove as part of the invention, including of course a chimericpolypeptide, as an active ingredient.

The invention is thus also a pharmaceutical composition for promotingbone growth, having a therapeutically effective amount of anypolypeptide described above as part of the invention.

The invention includes a method of increasing bone growth in a mammal byadministering a therapeutically effective amount of a polypeptide (or apharmaceutical composition including the polypeptide) described above aspart of the invention.

The invention includes the treatment of osteoporosis, promotion of bonegrowth in a mammal or treatment of a human of a bone reduction relateddisease.

The invention includes the use of a polypeptide having a sequenceaccording to any polypeptide of the invention in the preparation of amedicament for use in promoting bone growth or the treatment ofosteoporosis, etc.

The invention includes a diagnostic kit for determining the presence ofa polypeptide of the invention, in which the kit includes an antibody toa polypeptide (or polypeptides) linked to a reporter system wherein thereporter system produces a detectable response when a predeterminedamount of the polypeptide (or polypeptides) and the antibody becomebound together.

The invention includes an antibody which binds to a polypeptide of theinvention. Particularly, the invention includes an antibody which bindsto such a polypeptide when the antibody is synthesized using thepolypeptide.

The invention includes molecules, such as isolated nucleotide sequencesrelated to polypeptides of the invention. For example, the inventionincludes an isolated DNA fragment which encodes the expression of any ofthe polypeptides of the invention. It is of course understood that suchfragments can vary from one another due to the degeneracy of the geneticcode. Further, the invention includes a vector that has incorporatedinto it any such DNA sequence.

The invention includes an isolated DNA sequence encoding any amino acidsequence of the invention, or an analogue thereof, wherein the aminoacids in the sequence may be substituted, deleted or added, so long asbone stimulatory activity in mammals derived from the three dimensionalconformation of the sequence is preserved in a polypeptide having theamino acid sequence; sequences which hybridize to the DNA and encode anamino acid sequence of a polypeptide which displays bone stimulatoryactivity in mammals; and and DNA which differs from the sequence due tothe degeneracy of the genetic code.

The invention thus includes processes of producing any polypeptide ofthe invention, including a process which includes: a) preparing a DNAfragment containing a nucleotide sequence that encodes such apolypeptide; b) incorporating the DNA fragment into an expression vectorto obtain a recombinant DNA fragment which contains the DNA fragment andis capable of undergoing replication; c) transforming a host cell withthe recombinant DNA fragment to isolate a transformant which can expressthe polypeptide; and d) culturing the transformant to allow thetransformant to produce the polypeptide and recovering the polypeptidefrom resulting cultured mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following description, reference is made to accompanyingdrawings, wherein,

FIG. 1 graphically depicts the bone mineral apposition rate (μm per day)in rats provided with the chemically synthesized human N-acetyl(N-terminus) polypeptide (SEQ ID NO:2) through implantation inparathyroidectomized rats. The error bars indicate ±1 standard deviation(S.D.). The value of p was less than 0.001.

FIG. 2 graphically depicts right femoral bone calcium density of ratstreated over a four week period. Group A rats were ovariectomized andinjected daily with the chemically synthesized normal peptide (SEQ IDNO:1). Group B rats were ovariectomized and injected daily with controlsolution. Group C rats were subject to sham ovariectomization operationsand injected daily with control solution. Group D were intact ratsinjected daily with control solution. The error bars indicate ±1standard deviation (S.D.).

FIG. 3 graphically depicts the bone mineral apposition rate of rats asdetermined by tetracycline labeling after treatment as described inconnection with FIG. 2. The error bars indicate ±1 standard deviation(S.D.).

FIG. 4 graphically depicts femoral bone calcium concentration of ratstreated over an eight week period. Group A rats were ovariectomized andinjected daily with the chemically synthesized normal peptide (SEQ IDNO:1) beginning eight weeks after the operation. Group B rats weresimilarly ovariectomized and injected daily with control solution. GroupC rats were subject to sham ovariectomization operations and injecteddaily with control solution. Group D were intact rats injected dailywith control solution. The error bars indicate ±1 standard deviation(S.D.).

FIG. 5 graphically depicts the bone mineral apposition rate of intactrats as determined by tetracycline labeling. Group A rats were treatedwith rabbit antibodies to the chemically synthesized normal polypeptide(SEQ ID NO:1). Group B rats were treated with the same antibodies andthe polypeptide itself. Group C is the control group. The error barsindicate ±1 standard deviation (S.D.).

FIG. 6 shows a tricine SDS electrophoretic gel of the human chemicallysynthesized polypeptide (SEQ ID NO:1) and the same polypeptidecontaining a cys→ala substitution (SEQ ID NO:3).

FIG. 7 graphically depicts the bone mineral apposition rate (μm per day)in rats injected with the chemically synthesized human polypeptide (SEQID NO:1), Group A; the modified chemically synthesized human polypeptide(SEQ ID NO:3), Group B; and control, Group C. (N=6 for all groups). Theerror bars indicate ±1 standard deviation (S.D.).

FIG. 8 graphically depicts the bone mineral apposition rate (μm per day)in rats injected with N-terminus chemically synthesized polypeptides:SEQ ID NO:1 (Group A); SEQ ID NO:7 (Group B); SEQ ID NO:6 (Group C); SEQID NO:5 (Group D); and SEQ ID NO:4 (Group E). (N=6 for all groups). Theerror bars indicate ±1 standard deviation (S.D.).

FIG. 9 graphically depicts the bone mineral apposition rate (μm per day)in rats injection with chemically synthesized polypeptides: SEQ ID NO:8(Group G); SEQ ID NO:9 (Group H).

FIG. 10 is a DEXA image of a right femur of a rat showing scanned areas:A, proximal end; B, diaphysis; and C, distal end .

FIG. 11 is a DEXA image of a right femur of a rat showing scanned neckarea.

FIG. 12 graphically depicts the bone mineral apposition rate (μm perday) in rats injected with non-N-terminus chemically synthesizedpolypeptide fragments SEQ ID NO:1 (Group H); SEQ ID NO:16 (Group I); SEQID NO:15 (Group J); SEQ ID NO:14 (Group K); and SEQ ID NOS:10,11,12 & 13(Group L). (N=6 for all groups). The error bars indicate ±1 standarddeviation (S.D.).

FIG. 13 graphically depicts the bone mineral apposition rate (μm perday) in rats injected with chemically synthesized polypeptide fragmentsSEQ ID NO:1 (Group N); SEQ ID NO:7 (Group O); SEQ ID NO:9 (Group P); andSEQ ID NO:24 (Group Q), and a control group (Group M). The error barsindicate ±1 standard deviation (S.D.).

FIG. 14 graphically depicts the bone mineral apposition rate (μm perday) in rats injected with chemically synthesized polypeptide fragmentsSEQ ID NO:25 (Group S); SEQ ID NO:26 (Group T); SEQ ID NO:27 (Group U);SEQ ID NO:24 (Group V) and a control group (Group R). The error barsindicate ±1 standard deviation (S.D.).

FIG. 15 illustrates the amino sequences of the various polypeptidestested, active polypeptides being shown above the mid-line and sequenceswhich were not found to stimulate bone growth being below the mid-line.

METHODOLOGY

The applicable methodology as described in the General Methodologysection of international patent application No. PCT/CA 94/00144 wasfollowed here.

TOXICITY EXPERIMENTS INVOLVING N-TERMINAL ACETYL CHEMICALLY SYNTHESIZEDPOLYPEPTIDE (SEQ ID NO: 2)

A miniosmotic pump (Alzet) was loaded with about 1.5 ml of thechemically synthesized peptide having an N-terminal acetyl group (SEQ IDNO:2) in 0.1% acetic acid so as to give a calculated daily delivery ofabout 25 μg per day. A pump was implanted under the subcutaneous fasciaof the dorsal aspect of the left side of the thorax of five rats whichhad been parathyroidectomized seven days earlier. Five similarlyparathyroidectomized rats received similar implants containing only 0.1%acetic acid. Five intact rats were also used as controls.

Twenty-eight days later 0.5 ml of an aqueous solution of tetracyclinehydrochloride was injected intramuscularly into the right gluteusmaximus of each of the implanted rats, as described previously. Another48 hours later, a second injection of tetracycline hydrochloridesolution was injected. The rats were sacrificed another 24 hours later.

The bone mineral apposition rate was determined by examination of across-section of the lower metaphysis of the right femur of each of theten rats which had been given implants. The results are summarized inTable One depicted graphically in FIG. 1.

                  TABLE ONE                                                       ______________________________________                                        Comparison of the Group Arithmetic                                            Means Among Groups                                                            ______________________________________                                                    Test Group Control Group                                          ______________________________________                                        Mean          1.27 μm/d 0.67 μm/d                                       S.D.          0.18 μm/d 0.08 μm/d                                       N             5            5                                                  ______________________________________                                                    t          d.f                                                    ______________________________________                                        Test Group vs 7.14         8                                                  Control Group                                                                 ______________________________________                                    

Histological evaluation of selected tissues of the five rats of each ofthe groups indicated in Table One were carried out microscopically. Noevidence of toxic lesions was found.

EXPERIMENTS INVOLVING OVARIECTOMIZED RATS AND THE NORMAL CHEMICALLYSYNTHESIZED POLYPEPTIDE (SEQ ID NO:1), ADMINISTRATION OVER A FOUR WEEKPERIOD

Ovariectomies were performed on six female Sprague-Dawley rats, eachsedated with 1 mg of sodium barbiturate I.P. Sham operations werecarried out a second group of six rats. The rats were given two weeks torecover from the operations.

The six ovariectomized rats were injected subcutaneously with 100 μl ofa 0.1% acetic acid solution containing 100 μg of the chemicallysynthesized peptide (SEQ ID NO:1) every 24 hours for 28 days. On day 25,a tetracycline hydrochloride solution was injected intramuscularly intoeach rat so as to give 24 mg per Kg of body weight, as describedpreviously. On day 27, a second dose of tetracycline hydrochloride wasinjected and the rats were sacrificed on the 28th day.

A second group of six ovariectomized rats, was similarly treated with a0.1% acetic acid solution containing no peptide over the same 28 dayperiod. A third group of six rats, each of which had undergone the shamoperation, was similarly treated with a 0.1% acetic acid solutioncontaining no peptide over the same 28 day period. A fourth group of sixintact rats was similarly treated with a 0.1% acetic acid solutioncontaining no peptide over the same 28 day period.

Postmortem blood was taken by cardiac puncture and serum frozen untilanalyzed. A full autopsy was performed on each rat. No ill effects wereobserved in the rats treated with the polypeptide.

Each of the right femurs was dissected out from its soft tissue, fixedfor two days, and X-rays taken at 70 kV for 1 min., 2 min., and 3 min.The 3 minute exposures gave the most satisfactory results. The bonedensities of the femurs from the second group of rats, theovariectomized rats not treated with the peptide, showed a visibly lowerbone density.

The right femur of each rat was decalcified separately. Thedecalcification fluid consisted of 10% formic acid (v/v) and 5% sodiumcitrate (w/v) at pH 3.0. Each bone was placed in 6 ml of thedecalcification fluid. The fluid was replaced after 4 days, again afteranother 4 days, again after another 2 days, and again after another 3days. After another 2 days, the decalcification fluid was removed andreplaced by deionized water, and the sample agitated for 2 days. Thewater changed after two days and again after another day. After anotherday, all of the fluid samples for each rat were combined and the finalvolume of each adjusted to 50 ml with deionized water.

The volume of each right femur was determined by determining the volumeof water displaced when the bone was immersed in water. The calciumconcentration of each sample was determined according to standardmethods and the calcium density of each bone calculated. The results aretabulated in Table Two and graphically depicted in FIG. 2. As can beseen, the bone calcium concentration measured for the ovariectomizedrats treated with the peptide (SEQ ID NO:1) appears to be normal, whilethe calcium concentration of the untreated ovariectomized rats isdepressed.

                  TABLE TWO                                                       ______________________________________                                        Right Femoral Calcium Concentration of Ovariectomized Rats                    ______________________________________                                                Group A Group B   Group C   Group D                                   ______________________________________                                        Mean (μmol/ml)                                                                       7.57      6.61      7.45    7.69                                    N         6         6         6       6                                       S.D.      0.38      0.29      0.28    0.31                                    ______________________________________                                        GROUP     t             d.f.  p                                               ______________________________________                                        A vs B    4.90          10    <0.001                                          A vs C    0.62          10    >0.5                                            A vs D    0.60          10    >0.5                                            B vs C    5.08          10    <0.001                                          B vs D    6.20          10    <0.001                                          C vs D    1.40          10    >0.1                                            ______________________________________                                    

The bone mineral apposition rate was determined, as describedpreviously, by measurement of the lower metaphysis of the left femur.The results are tabulated in Table Three and graphically depicted inFIG. 3.

                  TABLE THREE                                                     ______________________________________                                        Bone Mineral Apposition Rates of Ovariectomized Rats                          ______________________________________                                               Group A Group B   Group C   Group D                                    ______________________________________                                        Mean (μm/day)                                                                       0.90      0.59      0.85    0.86                                     N        6         6         6       6                                        S.D.     0.12      0.07      0.07    0.09                                     ______________________________________                                        GROUP     t             d.f.  p                                               ______________________________________                                        A vs B    5.39          10    <0.001                                          A vs C    0.87          10    >0.5                                            A vs D    0.21          10    >0.5                                            B vs C    6.29          10    <0.001                                          B vs D    5.93          10    <0.001                                          C vs D    0.21          10    >0.5                                            ______________________________________                                    

EXPERIMENTS INVOLVING OVARIECTOMIZED RATS AND THE NORMAL CHEMICALLYSYNTHESIZED POLYPEPTIDE, ADMINISTRATION OVER AN EIGHT WEEK PERIOD

Eight weeks after ovariectomization, five ovariectomized rats wereinjected subcutaneously with 100 μl of a 0.1% acetic acid solutioncontaining 100 μg of the chemically synthesized peptide in which theN-terminal amino group was modified with an acetyl group (SEQ ID NO:2).This was done every 24 hours for eight weeks. On day 54, a tetracyclinehydrochloride solution was injected intramuscularly into the rightgluteus maximus of each rat so as to give 24 mg per Kg of body weight,as described previously. On day 56, a second dose of tetracyclinehydrochloride was injected and the rats were sacrificed on the 57th day.

A second group of seven ovariectomized rats, was similarly treated witha 0.1% acetic acid solution containing no peptide over the same period.A third group of five rats, each of which had undergone the shamoperation, was similarly treated with a 0.1% acetic acid solutioncontaining no peptide over the same period. A fourth group of fiveintact rats was similarly treated with a 0.1% acetic acid solutioncontaining no peptide over the same 8 week period. Two rats of thesecond group became ill during the 8 week period and were sacrificedprematurely.

Postmortem blood was taken by cardiac puncture and serum frozen untilanalyzed. An autopsy was performed on each rat. No obvious pathology wasobserved in the rats except for surgical scars and atrophy of the uterusand vagina of ovariectomized rats.

The right femurs were decalcified and calcium density determined asbefore. The results are presented in Table Four and FIG. 4.

                  TABLE FOUR                                                      ______________________________________                                        Right Femoral Calcium Concentration of Ovariectomized Rats                    ______________________________________                                                Group A Group B   Group C   Group D                                   ______________________________________                                        Mean (μmol/ml)                                                                       7.37      6.89      7.69    7.87                                    N         5         5         5       5                                       S.D.      0.15      0.32      0.30    0.24                                    ______________________________________                                        GROUP     t             d.f.  p                                               ______________________________________                                        A vs B    3.85          6     <0.005                                          A vs C    1.17          6     >0.2                                            A vs D    3.01          6     <0.01                                           B vs C    4.03          6     <0.005                                          B vs D    5.41          6     <0.001                                          C vs D    1.60          6     >0.1                                            ______________________________________                                    

SYNTHESIS OF ANTIBODIES TO CHEMICALLY SYNTHESIZED PROTEIN (SEQ ID NO: 1)

The chemically synthesized protein (SEQ ID NO:1) was coupled to KLH(keyhole limpet hemocyanin) with three different cross-linkers, asdescribed below.

GLUTARALDEHYDE COUPLING

In 2.5 ml of a PBS solution made up of 2.7 mM KCl, 1.2 mM KH₂ PO₄, 138mM NaCl, 8.1 mM Na₂ HPO₄, were diluted 5 mg of the peptide (SEQ ID NO:1)to obtain a final peptide concentration of 2 mg/ml. 10 mg of KLH werediluted in 5.0 ml PBS to obtain a final concentration of 2 mg/ml. To1.25 ml of the KLH solution were added 1.25 ml of the peptide solution.Glutaraldehyde was added to a final concentration of 0.25%. Theresultant solution was stirred for 1 hour at room temperature. Afterstirring, the solution was dialysed against 1 liter of PBS. The PBS waschanged three times.

CARBODIIMIDE (EDC) COUPLING

Peptide and KLH solutions were prepared as described in the precedingsection. To 1.25 ml KLH solution were added 1.25 ml peptide solution. Tothe resultant solution were added 2.5 mg of EDC. The solution wasstirred constantly at room temperature for 4 hours and then dialysedagainst 1 liter of PBS. The PBS was changed three times.

M-MALEIMIDOBENZOYL-N-HYDROXYSUCCINIMIDE ESTER (MBS) COUPLING

To 500 μl of H₂ O were added 5 mg of the peptide and the pH was adjustedto 8.5 with NaOH, to obtain a final concentration of 10 mg/ml.Citraconic anhydride was diluted in H₂ O to a concentration of 10 mg/ml.500 μl of the anhydride solution were added to the peptide solution 100μl at a time with adjustment of the pH to 8.5 between each addition. Thesolution was then stirred constantly at room temperature for 1 hour.This was followed by the addition of 100 μl of 1M sodium phosphatebuffer (pH 7.2) and then 900 μl of 100 mM sodium phosphate buffer (pH7.2). Sulfo-MBS was diluted in H₂ O to a concentration of 25 mg/ml and400 μl of this solution were added to the peptide solution to obtain anMBS concentration of about 5 mg/ml. This solution was stirred constantlyat room temperature for 30 minutes. 6 μl of β-mercaptoethanol were addedfor a final β-mercaptoethanol concentration of 35 mM. The solution wasstirred constantly at room temperature for 1 hour. KLH was dissolved inPBS at 3 mg/ml and 2.5 ml were added to the peptide solution. Thesolution was stirred constantly at room temperature for 3 hours and thendialysed against 1 liter of PBS, with three changes of the PBS. Thefinal peptide concentration was about 1 mg/ml and the final KLHconcentration was about 1.5 mg/ml.

ANTIBODY GENERATION

Rabbits were injected with the synthetic peptide solutions as follows.250 μl each of the glutaraldehyde- and EDC-coupled peptide solutionswere together mixed with 500 μl of Freund's adjuvant. This solution wasinjected intramuscularly into the rear legs of a rabbit, 500 μl per leg.The total amount of injected peptide was 0.5 mg. 500 μl of the syntheticpeptide coupled to KLH with MBS were mixed with 500 μl of Freund'sadjuvant. This solution was injected intramuscularly into the rear legsof another rabbit, 500 μl per leg. The total amount of injected peptidewas 0.5 mg.

The synthetic peptide was loaded onto two lanes, 1.5 μg and 4 μg, of agel (18% running, 5% stacking). The gel was blotted overnight at 30 Vand blocked with 3% milk in PBS. The gel was incubated overnight withrabbit serum diluted 1:250 in 1% milk/PBS followed by incubation withgoat anti-rabbit-alkaline phosphatase diluted 1:1000 for 1 hour. The gelwas then developed with substrate. The synthetic peptide was seen bycomasie blue staining. The peptide was detected by the second bleed ofeach rabbit and was not detected by the preimmune serum of eitherrabbit.

Interaction between immobilized peptide and serum antibodies was furtherstudied through surface plasmon resonance using BlAcore™. The syntheticpeptide was covalently immobilized on a dextran matrix by aminecoupling. Rabbit serum of different dilutions were injected over thesurface for five minutes and the amount of antibody bound to theimmobilized peptide determined. The titer is defined as the lastdilution of the serum giving a positive response, that is, greater than50 Resonance Units. Using this approach, antibodies were found to bepresent in serum from both rabbits and the interaction can be blocked bypreincubating the serum with the peptide. Antibodies in serum of therabbits were found not to interact with an immobilized unrelatedpeptide.

EXPERIMENTS INVOLVING RATS AND ANTIBODIES TO THE CHEMICALLY SYNTHESIZEDPEPTIDE

Antibody serum was prepared in 10 mM Tris.Cl at pH 7.4. Each of fiverats received 100 μl of the solution by injection into the left gluteusmaximus. Each rat of a second group of five rats was treated similarly,but with an additional injection of solution containing 45 μg of thepolypeptide (SEQ ID NO:1) into the right gluteus maximus. Each rat of athird group of five rats received an injection of 100 μl of 10 mMTris.Cl at pH 7.0.

Each of the fifteen rats was then injected as before with tetracyclinehydrochloride, in the amount of 24 mg per Kg of body weight. A seconddose of tetracycline hydrochloride was injected about 48 hours later.The rats were sacrificed after about another 24 hours.

The bone mineral apposition rate was determined by measurements,described above, of the lower right femoral metaphysis. The results aregiven in Table Five and FIG. 5.

                  TABLE FIVE                                                      ______________________________________                                        Bone Mineral Apposition Rates in Rats Injected with Antibody to the           Chemically Synthesized Peptide                                                ______________________________________                                                 Group A   Group B  Group C                                           ______________________________________                                        Mean (μm/day)                                                                         0.86        1.22     1.30                                          S.D.       0.02        0.08     0.11                                          N          5           5        5                                             ______________________________________                                                    t         d.f   p                                                 ______________________________________                                        Group A vs Group B                                                                          8.06        8     >0.2                                          Group A vs Group C                                                                          7.57        8     <0.001                                        Group B vs Group C                                                                          1.24        8     >0.2                                          ______________________________________                                    

Methodology and products can be thus be developed using antibody to thepolypeptide for use in detecting the polypeptide with which the antibodybinds. For example, antibody can be linked to or conjugated with any ofseveral well known reporter systems set up to indicate positivelybinding of the polypeptide to the antibody. Well known reporter systemsinclude radioimmuno assays (RIAs) or immunoradiometric assays (IRMAs).Alternatively, an enzyme-linked immunosorbent assay (ELISA) would havein common with RIAs and IRMAs a relatively high degree of sensitivity,but would generally not rely upon the use of radioisotopes. A visuallydetectable substance may be produced or at least one detectable in aspectrophotometer. An assay relying upon fluorescence of a substancebound by the enzyme being assayed could be used. It will be appreciatedthat there are a number of reporter systems which may be used, accordingto the present invention, to detect the presence of a particularpolypeptide. With standardized sample collection and treatment,polypeptide presence above a threshold amount in blood serum could wellbe determined.

Such a method based on antigenic response to the chemically synthesizedhuman polypeptide (SEQ ID NO:1) could be developed and variants of thepolypeptide obtained, as described above for amino acid substitution,deletion and addition, (and conjugates) could then be pre-screened aspotential bone stimulating factors. Those that react positively with theantibody to the already known peptide could then be tested for bonestimulatory effects in vivo using the system described herein for rats,for example.

Such an antibody-linked reporter system could be used in a method fordetermining whether blood serum of a subject contains a deficient amountof the polypeptide. Given a normal threshold concentration of such apolypeptide in blood serum of a given type of subject, test kits couldthus be developed.

EXPERIMENTS INVOLVING CHEMICALLY SYNTHESIZED HUMAN POLYPEPTIDECONTAINING CYSTEINE→ALANINE SUBSTITUTION

A modified sequence (SEQ ID NO:3) of the chemically synthesized peptide(SEQ ID NO:1) obtained by substitution of the cysteine residue atposition 13 by alanine was prepared by standard chemical procedures. Analanine residue is sterically similar to a reduced cysteine residuewhile rendering the polypeptide incapable of spontaneous dimerization. Atricine SDS electrophoretic gel of the modified and unmodified (normal)peptides is shown in FIG. 6.

Experiments were carried out on three groups of six rats weighingbetween 295 and 320 g. Al mg per ml solution of the modified peptide(SEQ ID NO:3) was prepared in 0.1% acetic acid. A 1 mg per ml solutionof the normal peptide (SEQ ID NO:1) was prepared in 0.1% acetic acid.Each rat of a first of the groups had subcutaneously injected into itsright thigh 0.1 ml of the modified peptide solution. Similarly, each ratof the second group was injected with 0.1 ml of the normal peptidesolution. Each rat of the third group, the control group, was injectedwith 0.1 ml of 0.1% acetic acid solution. Immediately following theseinjections, each rat was injected intramuscularly with 24 mg per Kg bodyweight of tetracycline hydrochloride dissolved in 0.5 ml of water. Asecond dose of tetracycline hydrochloride was administered 48 hourslater. The animals were sacrificed 24 hours after the second dose by CO₂narcosis. The lower metaphysis of the right femur was dissected out andfixed in a 10% aqueous solution of formaldehyde buffered at pH 7.2 byacetate buffer. Bone sections were prepared for measurement as describedabove.

The results are tabulated in Table Six and graphically depicted in FIG.7. As can be seen, the bone apposition rate for rats injected with themodified polypeptide is significantly greater than that for those of thecontrol group but below the bone apposition rate shown for the ratsinjected with the normal peptide.

                  TABLE SIX                                                       ______________________________________                                        Comparison of the Group Arithmetic Means Among Groups Injected with           Modified Peptide, Unmodified Peptide and Control                              ______________________________________                                                Group A  Group B    Control Group                                     ______________________________________                                        Mean      1.67 μm/d                                                                             1.35 μm/d                                                                             1.02 μm/d                                  S.D.      0.11 μm/d                                                                             0.16 μm/d                                                                             0.010 μm/d                                 N         6          6          6                                             ______________________________________                                                    t         d.f   p                                                 ______________________________________                                        Group A vs    12.2        10    <0.001                                        Control (Group C)                                                             Group B vs    4.69        10    <0.001                                        Control (Group C)                                                             Group A vs Group B                                                                          3.97        10    <0.005                                        ______________________________________                                    

EXPERIMENTS INVOLVING ACTIVE FRAGMENTS OF THE 36-AMINO ACID HUMANPOLYPEPTIDE

Polypeptides having the amino acid sequences identified as SEQ ID NOS:4,5, 6, 7, 8 and 9 were synthesized according to well known chemicalprocedures.

Sprague-Dawley rats were used as test animals to determine bone mineralapposition rate, as described above. Male rats having weights between280 and 380 g were subject to subcutaneous injection after one week ofacclimatization. Each animal was injected with 200 μl of a 0.1% aceticacid test solution, solutions having been prepared at concentrations toobtain a dosage of about 25 nmol of polypeptide per animal. Each testdose was immediately followed by intramuscular injection of 24 mg per Kgof body weight of tetracycline hydrochloride. A second injection oftetracycline was made 48 hours later.

Control Group: 0.1% acetic acid solution

Group A: SEQ ID NO:1:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gin Asn Gln Pro

Group E: SEQ ID NO:4:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val

Group D: SEQ ID NO:5:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu His Lys Lys Ala Ala

Group C: SEQ ID NO:6:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu

Group B: SEQ ID NO:7:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile

In a similar but separate set of experiments, bone mineral appositionrates were tested using the following chemically synthesizedpolypeptides:

Group F: SEQ ID NO:8:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys

Group G: SEQ ID NO:9:

Arg Thr Asn Glu His Thr Ala Asp Cys Lys

Bone mineral apposition rates were determined by measurements of thelower metaphysis of the right femur, as described previously. Resultsobtained in the two sets of experiments are summarized in Tables Sevenand Eight and graphically depicted in FIGS. 8 and 9. As can be seen, allof the polypeptides tested had a positive effect on bone appositionrate, i.e, displayed bone stimulatory activity.

                  TABLE SEVEN                                                     ______________________________________                                        Comparison of the Group Arithmetic Means Among First Groups                   Injected with Active Variants                                                 ______________________________________                                                                                  Con-                                Group A   Group B  Group C  Group D                                                                              Group E                                                                              trol                                ______________________________________                                        Mean 1.4      1.41     1.37   1.35   1.31   1.03                              S.D. 0.05     0.08     0.09   0.1    0.06   0.06                              N    6        6        6      6      6      6                                 ______________________________________                                                    t         d.f.  p                                                 ______________________________________                                        Group A vs Control                                                                          5.18        10    <0.001                                        Group B vs Control                                                                          9.67        10    <0.001                                        Group C vs Control                                                                          7.64        10    <0.001                                        Group D vs Control                                                                          6.92        10    <0.001                                        Group E vs Control                                                                          7.99        10    <0.001                                        Group A vs Group B                                                                          0.14        10    >0.5                                          Group A vs Group C                                                                          0.4         10    >0.5                                          Group A vs Group D                                                                          0.66        10    >0.5                                          Group A vs Group E                                                                          1.3         10    >0.2                                          Group B vs Group C                                                                          0.82        10    >0.4                                          Group B vs Group D                                                                          1.19        10    >0.2                                          Group B vs Group E                                                                          2.49        10    <0.05                                         ______________________________________                                    

                  TABLE EIGHT                                                     ______________________________________                                        Comparison of the Group Arithmetic Means Among Second Groups                  Injected with Active Variants                                                 ______________________________________                                                Group F  Group G    Control Group                                     ______________________________________                                        Mean      2.09 μm/d                                                                             2.83 μm/d                                                                             1.63 μm/d                                  S.D.      0.34 μm/d                                                                             0.19 μm/d                                                                             0.13 μm/d                                  N         4          3          4                                             ______________________________________                                                    t        d.f   p                                                  ______________________________________                                        Group F vs Control       6     0.047                                          Group G vs Control       5      0.0002                                        Group F vs Group G       5     0.215                                          ______________________________________                                    

BONE CALCIUM CONTENT EXPERIMENTS INVOLVING SEQ ID NO:7

A further set of experiments was conducted using the polypeptideidentified as SEQ ID NO:7 to determine the effect of the polypeptide onbone calcium content when administered to rats.

Ovariectomies were performed on rats as described above. A 0.1% aceticacid solution containing 25 nmoles of the polypeptide was administeredsubcutaneously to each rat each day for the duration of the experiment.One group of rats was treated for 12 weeks beginning 100 days afterovariectomization. Another group of rats was treated for eight weeksbeginning eight weeks after ovariectomization. Rats were sacrificed atthe end of the treatment period and dissected and post mortem assessmentof bone mineral content was carried out.

The lumbar spines L1-L4 were cleaned with a power nylon brush to removethe attached muscle. They were placed ventral side down under 3 cm ofdistilled water in a polypropylene container and scanned by a dualenergy x-ray absorptometer (DEXA), Hologic 100, to determine the calciumcontent in grams. The right femur of each rat was also dissected outintact and cleared of the attached muscles with a power nylon brush. Itwas scanned dorsal side down under 3 cm of distilled water by DEXA. Fourregions of the femur were scanned, as indicated in FIGS. 10 and 11: A,proximal end; B, diaphysis; C, distal end; and D, neck. The bone mineral(i.e., calcium) content in grams was estimated in the four zones of thefemur based on absorption and using an internal standard of the machine.

Results are tabulated in Tables Nine to Eighteen.

                  TABLE NINE                                                      ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected with               Polypeptide SEQ ID NO:7 Administered over 100 Days to                         Ovariectomized Rats-Bone Mineral Content Measured in Proximal End             of Femur                                                                      ______________________________________                                                     A-Ovariectomized                                                                            B-Ovariectomized                                   Control      (no polypeptide)                                                                            (with polypeptide)                                 ______________________________________                                        Mean (g.)                                                                             0.1503   0.1351        0.1411                                         S.D.    0.0159   0.0105        0.0155                                         N       14       14            11                                             ______________________________________                                                    t          d.f   p                                                ______________________________________                                        Control vs A  2.9772       26    <0.025                                       Control vs B  1.44         23    N.S.                                         Group A vs Group B                                                                          1.1634       23    N.S.                                         ______________________________________                                    

                  TABLE TEN                                                       ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected with               Polypeptide SEQ ID NO:7 Administered over 56 Days to                          Ovariectomized Rats-Bone Mineral Content Measured in Proximal End             of Femur                                                                      ______________________________________                                                           A-Ovariectomized                                                                           B-Ovariectomized                              Control    Sham    (no polypeptide)                                                                           (with polypeptide)                            ______________________________________                                        Mean (g.)                                                                            0.1451  0.1387  0.1368     0.1328                                      S.D.   0.0183  0.0166  0.028      0.0141                                      N      5       5       6          6                                           ______________________________________                                                   t          d.f    p                                                ______________________________________                                        Control vs Sham                                                                            0.7372       8      N.S.                                         Control vs A 0.6261       9      N.S.                                         Control vs B 1.6223       9      N.S.                                         Sham vs A    0.133        9      N.S.                                         Sham vs B    1.6229       9      N.S.                                         Group A vs B 0.3116       10     N.S.                                         ______________________________________                                    

                  TABLE ELEVEN                                                    ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 100 Days to                    Ovariectomized Rats-Bone Mineral Content Measured in Spine                    (L1-L4)                                                                       ______________________________________                                                     A-Ovariectomized                                                                            B-Ovariectomized                                   Control      (no polypeptide)                                                                            (with polypeptide)                                 ______________________________________                                        Mean (g)                                                                              0.5437   0.4364        0.4758                                         S.D.    0.0161   0.0089        0.0188                                         N       14       14            10                                             ______________________________________                                                    t          d.f   p                                                ______________________________________                                        Control vs A  5.8384       26    <0.001                                       Control vs B  2.7434       22    <0.0025                                      Group A vs Group B                                                                          2.0756       22    0.05                                         ______________________________________                                    

                  TABLE TWELVE                                                    ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 56 Days to                     Ovariectomized Rats-Bone Mineral Content Measured in Spine                    (L1-L4)                                                                       ______________________________________                                                           A-Ovariectomized                                                                           B-Ovariectomized                              Control    Sham    (no polypeptide)                                                                           (with polypeptide)                            ______________________________________                                        Mean (g.)                                                                            0.5542  0.5321  0.4322     0.4606                                      S.D.   0.0275  0.0172  0.0226     0.0234                                      N      5       5       6          6                                           ______________________________________                                                  t          d.f    p                                                 ______________________________________                                        Control vs Sham                                                                           0.6805       8      N.S.                                          Control vs A                                                                              4.4196       9      <0.005                                        Control vs B                                                                              3.1042       9      <0.025                                        Sham vs A   2.8382       9      <0.025                                        Sham vs B   1.9951       9      N.S.                                          Group A vs B                                                                              0.8759       10     N.S.                                          ______________________________________                                    

                  TABLE THIRTEEN                                                  ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 100 Days to                    Ovariectomized Rats-Bone Mineral Content Measured in                          Femoral Diaphysis                                                             ______________________________________                                                     A-Ovariectomized                                                                            B-Ovariectomized                                   Control      (no polypeptide)                                                                            (with polypeptide)                                 ______________________________________                                        Mean (g.)                                                                             0.2258   0.2146        0.2347                                         S.D.    0.0261   0.0106        0.0215                                         N       14       14            11                                             ______________________________________                                                    t          d.f   p                                                ______________________________________                                        Control vs A  0.8301       26    N.S.                                         Control vs B  0.9078       23    N.S.                                         Group A vs Group B                                                                          2.3079       23    <0.05                                        ______________________________________                                    

                  TABLE FOURTEEN                                                  ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 56 Days to                     Ovariectomized Rats-Bone Mineral Content Measured in                          Femoral Diaphysis                                                             ______________________________________                                                           A-Ovariectomized                                                                           B-Ovariectomized                              Control    Sham    (no polypeptide)                                                                           (with polypeptide)                            ______________________________________                                        Mean (g.)                                                                            0.2179  0.1918  0.1716     0.2091                                      S.D.   0.0156  0.0162  0.0272     0.0121                                      N      5       5       6          6                                           ______________________________________                                                  t          d.f    p                                                 ______________________________________                                        Control vs Sham                                                                           2.259        8      <0.05                                         Control vs A                                                                              3.3549       9      <0.025                                        Control vs B                                                                              1.9209       9      N.S.                                          Sham vs A   1.4571       9      N.S.                                          Sham vs B   1.1778       9      N.S.                                          Group A vs B                                                                              2.4926       10     <0.05                                         ______________________________________                                    

                  TABLE FIFTEEN                                                   ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 100 Days to                    Ovariectomized Rats-Bone Mineral Content Measured in Distal                   End of Femur                                                                  ______________________________________                                                     A-Ovariectomized                                                                            B-Ovariectomized                                   Control      (no polypeptide)                                                                            (with polypeptide)                                 ______________________________________                                        Mean (g.)                                                                             0.1597   0.1396        0.1424                                         S.D.    0.0185   0.0068        0.0132                                         N       14       14            11                                             ______________________________________                                                    t          d.f   p                                                ______________________________________                                        Control vs A  3.8255       26    <0.001                                       Control vs B  2.616        23    <0.025                                       Group A vs Group B                                                                          0.6984       23    N.S.                                         ______________________________________                                    

                  TABLE SIXTEEN                                                   ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 56 Days to                     Ovariectomized Rats-Bone Mineral Content Measured in Distal                   End of Femur                                                                  ______________________________________                                                           A-Ovariectomized                                                                           B-Ovariectomized                              Control    Sham    (no polypeptide)                                                                           (with polypeptide)                            ______________________________________                                        Mean (g.)                                                                            0.1826  0.154   0.1304     0.1347                                      S.D.   0.0122  0.0118  0.0094     0.0039                                      N      5       5       6          6                                           ______________________________________                                                  t          d.f    p                                                 ______________________________________                                        Control vs Sham                                                                           3.7549       8      <0.025                                        Control vs A                                                                              8.0183       9      <0.001                                        Control vs B                                                                              9.1462       9      <0.001                                        Sham vs A   3.7046       9      <0.005                                        Sham vs B   3.8149       9      <0.005                                        Group A vs B                                                                              1.0274       10     N.S.                                          ______________________________________                                    

                  TABLE SEVENTEEN                                                 ______________________________________                                        Comparison of Group Arithmetic Means Among Groups                             Injected with Polypeptide SEQ ID NO:7 Administered over 100                   Days to Ovariectomized Rats-Bone Mineral Content Measured                     in Femoral Neck                                                               ______________________________________                                                     A-Ovariectomized                                                                            B-Ovariectomized                                   Control      (no polypeptide)                                                                            (with polypeptide)                                 ______________________________________                                        Mean (g.)                                                                             0.0334   0.0303        0.0351                                         S.D.    0.0049   0.004         0.0031                                         N       14       14            10                                             ______________________________________                                                    t         d.f   p                                                 ______________________________________                                        Control vs A  1.3978      26    N.S.                                          Control vs B  1.0326      21    N.S.                                          Group A vs Group B                                                                          2.259       21    P < 0.005                                     ______________________________________                                    

                  TABLE EIGHTEEN                                                  ______________________________________                                        Comparison of Group Arithmetic Means Among Groups Injected                    with Polypeptide SEQ ID NO:7 Administered over 56 Days to                     Ovariectomized Rats-Bone Mineral Content Measured in                          Femoral Neck                                                                  ______________________________________                                                           A-Ovariectomized                                                                           B-Ovariectomized                              Control    Sham    (no polypeptide)                                                                           (with polypeptide)                            ______________________________________                                        Mean (g.)                                                                            0.0277  0.0255  0.0202     0.0274                                      S.D.   0.002   0.0038  0.0028     0.0013                                      N      5       5       6          6                                           ______________________________________                                                  t          d.f    p                                                 ______________________________________                                        Control vs Sham                                                                           1.1534       8      N.S.                                          Control vs A                                                                              4.9809       9      <0.001                                        Control vs B                                                                              0.3342       9      N.S.                                          Sham vs A   2.662        9      <0.05                                         Sham vs B   1.1462       9      N.S.                                          Group A vs B                                                                              5.6713       10     <0.005                                        ______________________________________                                    

As can be seen from the tabulated data, the increase in in vivo calciumbone content is most obvious in the femoral neck and femoral diaphysis,implying that the effect of the administered peptide can be sitespecific, possibly being greater at skeletal sites under mechanicalstress.

EXPERIMENTS INVOLVING OTHER FRAGMENTS OF THE 36-AMINO ACID HUMANPOLYPEPTIDE

Polypeptide fragments of the normal polypeptide (SEQ ID NO:1) were alsosynthesized and tested for bone stimulatory activity as with theC-terminus fragments.

Control Group: 0.1% acetic acid

Group H: SEQ ID NO:1:

Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro AsnThr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Pro

Group I: SEQ ID NO:16:

Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile

Group J: SEQ ID NO:15:

Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His LysLys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn

Group K: SEQ ID NO:14:

Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu ThrLeu Met Val Leu Asp

Group L: SEQ ID NOS: 10,11,12 & 13 (mixture):

Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Leu HisLys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Leu His Lys Lys AlaAla Glu Thr Leu Met Val Leu Asp Gln Leu His Lys Lys Ala Ala Glu Thr LeuMet Val Leu Asp

Bone mineral apposition rates were again determined by measurement ofthe lower metaphysis of the right femur. Results obtained are summarizedin Table Nineteen and graphically depicted in FIG. 12. As can be seen inFIG. 12, none of the non-N-terminus variants identified as SEQ ID NO:10, 11, 12, 13, 14, 15 or 16 was found to increase the bone appositionrate with respect to the control.

                                      TABLE NINETEEN                              __________________________________________________________________________    Summary of the Group Arithmetic Means for Bone Apposition                     Rates of Rats Injected with Non-N-terminus Variants                                  Group H                                                                            Group I                                                                           Group J                                                                           Group K                                                                            Group L                                                                            Control                                         __________________________________________________________________________    Mean (μm/day)                                                                     1.5  1.02                                                                              0.92                                                                              0.92 0.98 1.02                                            S.D.   0.09 0.12                                                                              0.09                                                                              0.04 0.09 0.06                                            N      6    6   6   6    6    6                                               __________________________________________________________________________

A polypeptide having the ten amino acid sequence of SEQ ID NO:9, butprotected at both ends was synthesized and tested for bone stimulatoryactivity in comparison to polypeptides identified as SEQ ID NOS: 1, 7and 9. The protected polypeptide was acetylated at the amino terminusand amidated at the carboxy terminus and is identified herein as SEQ IDNO:24. Results obtained according to experimental procedures describedabove, using about 125 nmoles of polypeptide per Kg of body weight ofanimal are presented in Table Twenty and FIG. 13.

                  TABLE TWENTY                                                    ______________________________________                                        Comparison of the Mean Bone Apposition Rates Among Rats Treated               with SEQ ID NOs: 1, 7, 9 and 24                                               ______________________________________                                               SEQ ID    SEQ ID  SEQ ID  SEQ ID                                              NO:1      NO:7    NO:9    NO:24 Control                                ______________________________________                                        Mean   2.03      2.18    2.11    2.72  1.7                                    (μm/day)                                                                   S.D.   0.1       0.26    0.18    0.15  0.28                                   N      6         6       6       6     6                                      ______________________________________                                                        t          d.f.  p                                            ______________________________________                                        SEQ ID NO:1 vs Control                                                                        >2.6       10    <0.025                                       SEQ ID NO:7 vs Control                                                                        -3.03      10    <0.003                                       SEQ ID NO:9 vs Control                                                                        -2.96      10    <0.006                                       SEQ ID NO:24 vs Control                                                                       -7.73      10    <0.00                                        SEQ ID NO:1 vs 7                                                                              -1.28      10    >0.2                                         SEQ ID NO:1 vs 9                                                                              -0.898     10    >0.3                                         SEQ ID NO: vs 24                                                                              -4.42      10    <0.002                                       SEQ ID NO:7 vs 9                                                                              0.548      10    >0.5                                         SEQ ID NO:7 vs 24                                                                             -4.42      10    <0.002                                       SEQ ID NO:9 vs 24                                                                             -6.38      10    <0.00                                        ______________________________________                                    

There are literature reports that the presence of histidine and cysteineresidues in polypeptides [Int. J. Peptide Protein Res. 45, 1995,547,553]. The following analogues of the polypeptide identified as SEQID NO:9 were synthesized:

SEQ ID NO:25:

CH₃ CO--Arg Thr Asn Glu His Thr Ala Glu Cys Lys--NH₂

SEQ ID NO:26:

CH₃ CO--Arg Thr Gln Glu His Thr Ala Glu Cys Lys--NH₂

SEQ ID NO:27:

CH₃ CO--Arg Thr Gln Glu His Thr Ala Asp Cys Lys--NH₂

Each of the polypeptides having the sequences identified as SEQ ID NO: 7and 24 was dissolved in 5 mM acetic acid to a final concentration of 1mg/ml and incubated at 37° C. The peptide compositions were analyzedweekly by capillary electrophoresis on a P/ACE 6000 system (Beckman)using 50 mM sodium phosphate pH 2.5 as the running buffer on a 57 cmlong×75 μm internal diameter capillary. The incubated peptide (20 μl)was diluted with 80 μl of running buffer, placed in a 500 μl vial priorto injection onto the P/ACE using pressure for 20 seconds. Followingelectrophoresis of the incubated peptide at 30 kV for 15 min a secondrun was carried out using freshly dissolved peptide as a control.

Analytical results indicated that each of the polypeptides testedunderwent modification. Mass spectroscopic results (not shown) indicatedthat both peptides were breaking down to smaller fragments, i.e.,undergoing proteolysis.

Each of the polypeptides was dissolved in 20 mM sodium phosphate pH 3.0,20 mM ammonium acetate pH 4.0, 20 mM ammonium acetate pH 5.0, 20 mM MESpH 6.0, 20 mM sodium phosphate pH 7.0, 20 mM sodium phosphate pH 7.5, 20mM sodium phosphate pH 8.0, 20 mM ammonium acetate pH 8.5 or 20 mMammonium acetate pH 9.5 to a final concentration of 1 mg/ml. The peptidewas incubated at 37° C. and the peptide assayed weekly by separation onP/ACE as described above. Some samples were separated by RP-HPLC and theisolated peaks subjected to mass spectroscopic analysis.

Analytic results indicated that the polypeptide identified as SEQ ID NO:24 was most stable at a pH near 4.5. When incubated above pH 6.0, thepeptide dimerized. The peptide degraded when incubated below pH 4.0. Thepolypeptide having the amino acid sequence identified as SEQ ID NO:7 hada similar stability profile.

The protected polypeptide identified as SEQ ID NO:24 was dissolved in 20mM or 250 mM ammonium acetate pH 4.5 to a final concentration of 1mg/ml. In some experiments the buffer was supplemented with 20 mM EDTA.The peptide solution was then incubated at -70° C., -20° C., 4° C. orroom temperature (22° C). Samples were assayed weekly by electrophoresison the P/ACE as described above and/or by RP-HPLC.

Analytical results indicated that the peptide is very stable as a powderand when dissolved in pH 4.5 buffer. Modification of the peptideincubated at room temperature was observed after 7 days as a peakeluting before the intact peptide on the RP-HPLC chromatogram. Thepeptide modification was not altered by addition of 20 mM EDTA or byincubation in 250 mM ammonium acetate. Dissolved peptide incubated at 4°C., -20° C. or -70° C. were unchanged as compared with dry peptidestored at -70° C. After 14 days of incubation at room temperature, theHPLC profile included the two peaks observed at day 7, plus anadditional peak with a retention time greater than intact peptide. Whilethe dissolved peptide incubated at room temperature continued to becomemodified with increasing time, it was apparent that it was most stablein the lower salt buffer with the addition of 20 mM EDTA. Incubation ofpeptide at all other temperatures did not significantly alter the HPLCprofile as compared with dry peptide stored at -70° C. up to 28 days.

The stability of each of the polypeptides having the amino acidsequences identified as SEQ ID NOS: 25, 26 and 27 was tested asdescribed above.

The polypeptide having SEQ ID NO: 27 was found to be unstable at all pHstested when incubated at 37° C. for 3 days. Numerous additional peakswere observed on its electropherogram. The polypeptide having SEQ IDNO:25 was stable at pH 2.5-3.0 for 20 days. The polypeptide having SEQID NO:26 was found to be stable at pH 2.5-3.0 when incubated at 37° C.

In general, polypeptides having amino acid sequences identified as SEQID NOS: 7 and 24 degraded over time when dissolved in dilute acids.These peptides were found to be most stable when dissolved in pH 4.5buffer. Analogues, SEQ ID NOS: 25 and 26 were found to be more stablethan SEQ ID NOS: 7 or 24, while SEQ ID NO:27 was found to besignificantly less stable.

Polypeptide having the amino acid sequences identified as SEQ ID NOS:25, 26 and 27 were tested for bone stimulatory activity in comparison tothe polypeptide identified as SEQ ID NOS: 24. Results obtained accordingto experimental procedures described above are presented in TableTwenty-One and FIG. 14.

                  TABLE TWENTY-ONE                                                ______________________________________                                        Comparison of the Mean Bone Apposition Rates Among Rats                       Treated with SEQ ID NOs: 24, 25, 26 and 27                                           SEQ ID    SEQ ID  SEQ ID  SEQ ID                                              NO:24     NO:25   NO:26   NO:27 Control                                ______________________________________                                        Mean   2.04      1.94    2.88    1.72  1.36                                   (μm/day)                                                                   S.D.   0.14      0.23    0.47    0.33  0.14                                   N      4         4       4       4     4                                      ______________________________________                                    

A summary of the results obtained with respect to particular polypeptidesequences tested is provided in FIG. 15.

As can be seen, the polypeptide identified as SEQ ID NO:24 (and thecorresponding unprotected polypeptide, SEQ ID NO:9) has a sequence of 10amino acids contained in the 36 amino acid sequence of the polypeptideidentified as SEQ ID NO:1. Thus, in vivo bone stimulatory activity canbe retained with a polypeptide in which as little as 28% of the aminoacid sequence of SEQ ID NO:1 is conserved. In particular, the protectedversion of the 10-amino acid polypeptide sequence, SEQ ID NO:24, has abone stimulatory effect which exceeds that of either SEQ ID NO:1 or the10-amino acid unprotected version, SEQ ID NO:9.

Further, As can be seen the polypeptide identified as SEQ ID NO:26 (andthe corresponding unprotected polypeptide, SEQ ID NO:29) has a sequenceof 10 amino acids, only eight of which are identical to those containedin the 36 amino acid sequence of the polypeptide identified as SEQ IDNO:1. This indicates that in vivo bone stimulatory activity can beretained in a polypeptide in which as little as 22% of the amino acidsequence of SEQ ID NO:1 is conserved. On a molar basis, the protectedsequence (SEQ ID NO:26) has been found, at least under the conditions ofthe foregoing experiments, have an even more potent effect than that ofSEQ ID NO:24.

Likewise, the polypeptide identified as SEQ ID NO:25 (and thecorresponding unprotected polypeptide, SEQ ID NO:28) has a sequence of10 amino acids, only nine of which are identical to those contained inthe 36 amino acid sequence of the polypeptide identified as SEQ ID NO:1.This indicates that in vivo bone stimulatory activity can be retained ina polypeptide in which as little as 25% of the amino acid sequence ofSEQ ID NO:1 is conserved. On a molar basis, the protected sequence (SEQID NO:25) has been found under the conditions of the experimentsdescribed above to have a comparable bone stimulatory effect to that ofSEQ ID NO:24.

The polypeptide identified as SEQ ID NO:27 (and the correspondingunprotected polypeptide, SEQ ID NO:30) has a sequence of 10 amino acids,only nine of which are the same as those contained in SEQ ID NO:1. Theprotected version of this polypeptide was found to have the bonestimulatory effect also, but it was not as great as that of SEQ IDNO:26.

As indicated in the experiments described above, the polypeptides varyfrom each other according to the conditions to which the polypeptidesare exposed. It is generally desirable that an active polypeptide not bedegraded to an inactive or less active moiety when stored or duringadministration. Information about the stability of an active fragment isuseful in formulating preparations for storage and administration.Stability information might also be useful in selecting a fragment thatis longer lived once administered to an individual.

Of course it is known to those skilled in the art that polypeptideswhich provide similar activity are generally related by having the sameor similar three-dimensional portion(s) which interacts with anotheragent, such as a receptor with which the polypeptide binds in some way.This is why it is possible to have several polypeptides that are relatedto each other that display similar bone-stimulating activity.

The present invention provides a synthetic polypeptide having in vivobone stimulatory activity in mammals and which increases calcium densityor content in bones of mammals, having an amino acid sequence which isat least about 19% conserved in relation to the amino acid sequenceidentified as SEQ ID NO:1 and having at least one amino acid deletedtherefrom, or a homologue thereof. In the context of this invention, apeptide containing an amino acid sequence that can be aligned with thatof SEQ ID NO:1 such that at least about 30% of individual amino acidresidues of the original sequence are present in the peptide is said tobe about 30% conserved with the amino acid sequence identified as SEQ IDNO:1, allowing for homologous substitutions and a limited number ofinsertions or deletions between aligned sequences. An amino acidsequence having seven out of the 36 amino acid residues of SEQ ID NO:1in aligned sequence would be 19% conserved. An amino acid sequencehaving eight out of the 36 amino acid residues of SEQ ID NO:1 in alignedsequence would be 22% conserved, such as is the case of SEQ ID NOS:26and 29. An amino acid sequence having nine out of the 36 amino acidresidues of SEQ ID NO:1 in aligned sequence would be 25% conserved, asfor SEQ ID NOS:25, 27, 28 and 30. An amino acid sequence having ten outof the 36 amino acid residues of SEQ ID NO:1 in aligned sequence is 28%conserved, as for SEQ ID NOS:9 and 24.

Described in a slightly different way, a polypeptide of the presentinvention is an amino acid sequence corresponding to SEQ ID NO:1 with(a) one amino acid to 4 amino acids deleted from the N-terminus of SEQID NO:1, (b) one to 22 amino acids deleted from the C-terminus of SEQ IDNO:1, or both (a) and (b); or a functionally equivalent homologue. Itmay be found possible to delete 5 or 6 or more amino acids from theN-terminus or to delete more than 22 amino acids from the C-terminus ofSEQ ID NO:1.

In another sense, the polypeptide of the present invention can bedescribed as a polypeptide exhibiting bone stimulatory activity inmammals, the polypeptide having the sequence identified as SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, or SEQ ID NO:9; analogues thereof wherein the aminoacids in the sequence may be substituted, deleted or added, so long asthe bone stimulatory activity in mammals derived from the threedimensional structure of the sequence is preserved; and conjugates ofeach of the polypeptides or analogues thereof, wherein if thepolypeptide sequence has that identified as SEQ ID NO:1, then there isat least one amino acid deleted therefrom.

A polypeptide of the present invention would include such a sequencewhich sequence would have a molecular weight in the range of from about1000 to 4000. It is to be understood however that the sequence might beadded to by conjugation or other technique, which could increase themolecular weight of the overall compound beyond 4000.

It will also be understood, without the intention of being limitedthereby, that a variety of substitutions of amino acids is possiblewhile "preserving" the three-dimensional structure responsible for thebone stimulatory effect of the polypeptides disclosed herein. It is thusexpected, for example, that interchange among non-polar aliphaticneutral amino acids, glycine, alanine, proline, valine and isoleucine,would be possible. Likewise, substitutions among the polar aliphaticneutral amino acids, serine, threonine, methionine, cysteine, asparagineand glutamine could possibly be made. This being said, the linkage ofthe peptides together by the disulfide bridge appears to be of someimportance, and so the lone cysteine residue should probably be heldintact and other amino acids capable of forming a disulfide linkage notbe substituted elsewhere in the sequence, although as seen above asuccessful cys→ala substitution was effected (SEQ ID NO:3).Substitutions among the charged acidic amino acids, aspartic acid andglutamic acid, can be made, as shown above, and substitutions among thecharged basic amino acids, lysine and arginine are also likely to bepossible. Substitutions can be made alone or in combination. These sortsof substitutions and interchanges are well known to those skilled in theart. U.S. Pat. Nos. 5,487,983 and 5,512,548, for instance, describesother possible substitutions including substitutions involving aminoacids not encoded by the gene. Other substitutions might well bepossible.

The importance of the N-terminus portion of the sequence is evident fromthe results described herein. The polypeptides (SEQ ID NOS:9, 24, 25,26, 27, 28, 29 and 30) having amino acids 5 to 14 of SEQ ID NO:1displays bone stimulatory activity while polypeptides lacking the firstnine N-terminus amino acids, but having amino acids 10 to 32 (SEQ IDNO:14) or amino acids 20 to 35 (SEQ ID NO:10) do not display bonestimulatory activity. It may be that it is possible to delete more aminoacids from either end of the polypeptide identified as SEQ ID NO:9 whileretaining the three-dimensional configuration of the subsequence of thepolypeptide responsible for bone stimulatory activity. Internaldeletions, although they might be possible to some limited extent,should be few. Of particular note, is the polypeptide having thesequence identified as SEQ ID NO:16, which differs by only one aminoacid residue from the amino acid sequence identified as SEQ ID NO: 9.The former does not display activity while the latter does display bonestimulatory activity. It is possible using the experimental methodsdisclosed herein to distinguish between sequences which do and do notstimulate bone growth and which do and do not increase calcium bonecontent.

It should still be possible for minor additions of amino acids to bemade at the ends of the sequence and symmetrical or nearly symmetricaladditions to the carboxy and amino terminals are likely to be possible.Internal additions, although likely to be possible to some limitedextent, should be few.

Of the above-listed modifications to the sequence, terminal additions,deletions or substitutions are most likely to be most useful, as such amodification can serve a variety of functions: an identifying group asfor use in a radioimmunoassay; or a linking group, as examples.

As with the normal peptide (SEQ ID NO:1), an active subsequencecontaining a cysteine residue (i.e., SEQ ID Nos: 4, 5, 6, 7, 8 or 9) canspontaneously dimerize and exist in the dimeric form, at least undercertain conditions.

A further advantage may be obtained through chimeric forms of theprotein, as known in the art. A DNA sequence encoding the entireprotein, or a portion of the protein, could thus be linked with asequence coding for the C-terminal portion of E. coli β-galactosidase toproduce a fusion protein, for example. An expression system for humanrespiratory syncytial virus glycoproteins F and G is described in U.S.Pat. No. 5,288,630, issued Feb. 22, 1994, and references cited therein,for example.

A polypeptide of the present invention would usually be synthetic,whether prepared by techniques of conventional "chemistry" or byrecombinant techniques. Here, a polypeptide so produced is referred toas being substantially pure or biochemically pure when it is generallyfree of polypeptides or proteins with which it would occur if founddirectly in nature, as in blood serum from an animal, for example.

Nucleic acid (DNA) sequences coding for the active portions of thenormal polypeptide would be as follows:

SEQ ID NO:17 (corresponding to SEQ ID NO:4):

GGG ATC GGA AAA CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA ATT AM CCG AACACC TTG CAT AAA AAA GCT GCA GAG ACT TTA ATG GTC

SEQ ID NO:18 (corresponding to SEQ ID NO:5):

GGG ATC GGA AAA CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA ATT AAA CCG AACACC TTG CAT AAA AAA GCT GCA

SEQ ID NO:19 (corresponding to SEQ ID NO:6):

GGG ATC GGA MA CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA ATT AAA CCG MCACC TTG

SEQ ID NO:20 (corresponding to SEQ ID NO:7):

GGG ATC GGA AAA CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA ATT

SEQ ID NO:21 (corresponding to SEQ ID NO:8):

GGG ATC GGA AAA CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA

SEQ ID NO:22 (corresponding to SEQ ID NO:9):

CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA

Hypothetical coding sequences for polypeptides based on SEQ ID NO:1 buthaving amino acid residues substituted therefor, are:

SEQ ID NO:31 (corresponding to SEQ ID NO:28):

CGA ACA MT GAA CAT ACG GCA GAA TGT AAA

SEQ ID NO:32 (corresponding to SEQ ID NO:29):

CGA ACA CAA GAA CAT ACG GCA GAA TGT AAA

SEQ ID NO:33 (corresponding to SEQ ID NO:30):

CGA ACA CAA GAA CAT ACG GCA GAT TGT AAA

Accordingly, a vector incorporating such a DNA sequence could beconstructed for use in synthesizing a polypeptide, as describedpreviously, and particularly in international patent application No.PCT/CA 94/00144. The DNA sequence coding for the polypeptide identifiedas SEQ ID NO:1 is given as SEQ ID NO:23 in the sequence listing of thisspecification.

A DNA sequence or fragment of the present invention may be any fragmentthat contains a nucleotide sequence which encodes a polypeptide of thepresent invention. In addition to any of the above coding sequences, theDNA fragment can have an appropriate promoter and an SD sequence (or asuitable ribosome binding site) at its 5'-end, and if necessary, anucleotide sequence containing a translation initiation codon at the5'-end and a nucleotide sequence containing a termination codon at the3'-end.

As known to those skilled in the art, the genetic code is "degenerate".A nucleotide in a gene sequence can thus be replaced by anothernucleotide in accordance with the degeneracy of a particular codon(coding triplet), without changing the amino acid sequence of thepolypeptide coded for by the gene. A DNA fragment of the presentinvention can thus be derived from any of the above sequences (and DNAsequences corresponding to substituted polypeptide or other analoguesnot explicitly illustrated), and such replacement might be done in sucha way that the resulting codon(s) shows a high utilization frequency ina specific host cell when producing a polypeptide of the presentinvention using genetic engineering techniques.

As used herein, "protected" terminal amino group refers to a terminalamino group (N-terminus) coupled with any of various amino4terminalprotecting groups that can be employed in peptide synthesis. Examples ofsuitable groups include acyl protecting groups, for example, formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups, for example benzyloxycarbonyl; andaliphatic urethane protecting groups, for example t-butoxycarbonyl oradamantyloxycarbonyl (Gross and Mienhofer, eds., The Peptides, vol 3,pp. 3 to 88 (Academic Press, New York, 1981)).

As used herein, "protected" terminal carboxyl group refers to a terminalcarboxyl group (C-terminus) coupled with any of various carboxy-terminalprotecting groups. As will be readily apparent to a person skilled inthe art, suitable groups include t-butyl, benzyl or other acceptablegroups linked to the terminal carboxyl group through an ester or etherbond.

Compounds within the scope of this invention can be synthesizedchemically by means well known in the art such, for example, solid phasepeptide synthesis. The synthesis is commenced from the carboxy-terminalend of the peptide using an α-amino protected amino acid.t-Butyloxycarbonyl (Boc) protective groups, or other suitable protectivegroups, can be used (Stewart et al., "Solid-Phase Peptide Synthesis," W.H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963); Vale etal., Science 213, 1394-1397 (1981), andMarke etal. J. Am Chem. Sci. 103, 3178 (1981)). Synthetic methods arealso described in "Principles of Peptide Synthesis" M. Bodansky Ed.(Spring-Verlag 1984). These and other methods of peptide synthesis arealso exemplified by U.S. Pat. Nos. 3,862,925, 3,842,067, 3,972,859,4,105,602, 4,683,291, 4,244,946 and 4,305,872.

Compounds may also be synthesized using manual or automatic techniques,for example, an Applied BioSystems 430A Peptide Synthesizer (FosterCity, Calif.) or a Biosearch SAM 11 automatic peptide synthesizer(Biosearch, Inc., San Rafael, Calif.).

Compounds of the present invention and compositions containing them finduse in numerous therapeutic and prophylactic applications in theprevention and treatment of bone reduction related to a disease.Compounds can thus be used as treatments to promote bone growth, in thetreatment of osteoporosis, for example, by any suitable route. Thepreferred routes are suitable for delivery of polypeptide-type compoundsto the bloodstream of a subject, bearing in mind proper storage andhandling conditions required for polypeptides such as those describedherein.

Thus the present invention also provides compositions containing aneffective amount of compounds of the present invention, including thenontoxic addition salts, amides and esters thereof, which may, alone,serve to provide the treatment benefits described above. Suchcompositions can also be provided together with physiologicallytolerable liquid, gel or solid diluents, adjuvants and excipients.

In the above examples involving subsequences of about 125 nmol ofpolypeptide per kg of bodyweight of animal was used per administration.In practice, particularly as human subjects are concerned, the dailydosage may well be between 0.01 and 300 mg or more per kg of bodyweight.More preferably, the dosage would be in the neighborhood of from about0.1 to about 30 mg per kg of bodyweight. It may be that the preferredfrequency of administration would be greater or less than once per day,depending upon the route of administration, convenience, and thevariation of effectiveness of treatment with frequency of and amountused per administration. The dosage administered also depends on thesubject and to which effect such administration is to give. The dosageof any one or more of the compounds will depend on many factorsincluding the specific compound or combination of compounds beingutilized, the mode of administration, and the mammal being treated.Dosages of a particular compound or combination of compounds can bedetermined using conventional considerations; for example, by customarycomparison of the differential activities of the subject compounds andthat of a known agent, that is, by means of an appropriatepharmacological protocol in which, for example, bone density of subjectsis measured over time.

Pharmaceutical preparations include any of the compounds prepared as aninjectable solution, including an injectable solution prepared justprior to use, for promoting bone growth and/or treatment ofosteoporosis. An injectable can be either a liquid solution orsuspension; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active polypeptide is often mixed with diluents andexcipients which are physiologically tolerable and compatible with thepolypeptide. Suitable diluents and excipients are, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired, the compositions can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, stabilizingor pH buffering agents, and the like.

Pharmaceutical preparations include the employment of the compounds inadmixture with conventional excipients, that is, pharmaceuticallyacceptable organic or inorganic carrier substances which do notdeleteriously react with the compounds, and which possibly enhance thestorage and handling stability of the compounds. The preparativeprocedure may include the sterilization of the pharmaceuticalpreparations. The compounds may be mixed with auxiliary agents such aslubricants, preservatives, stabilizers, salts for influencing osmoticpressure, etc., which do not react deleteriously with the compounds.

The compositions are conventionally administered parenterally, byinjection, for example either subcutaneously or intravenously.Additional formulations which are suitable for other modes ofadministration include suppositories, intranasal aerosols, and, in somecases, oral formulations. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills capsules, sustained release formulations, or powders, andcontain 10%-95% of active ingredient, preferably 25%-70%. These oralformulations include formulations designed to protect the peptide untilit can be absorbed.

The peptide compounds may be formulated into the compositions as neutralor salt forms. Pharmaceutically acceptable non-toxic salts include theacid addition salts (formed with the free amino groups) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups maybe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

The compounds of the invention can be homopolymerized to themselves(i.e., (peptide)_(n)) or, heteropolymerized to one another. Thecompounds can also be conjugated to biocompatible polymeric compounds,such as BIOPOL™ (WR Grace & Co.-Conn.).

If prepared using recombinant techniques, a DNA sequence encoding adesired polypeptide of the present invention is synthesized usingstandard automated techniques, or the coding sequences or portionsthereof are retrieved from cDNA or genomic libraries. This DNA isligated into suitable expression vectors and these vectors aretransformed into appropriate hosts. A variety of expression vector/hostcell systems can be used, including both procaryotic and eukaryoticculture systems.

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication origins, and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species (Bolivar et al., (1977) Gene 2:95. Commonly usedprocaryotic control sequences, which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta-lactamase (penicillinase), lactose (Inc) promotersystems (Chang et al., (1977) Nature 198:1056), the tryptophan (trp)promoters system (Goeddel et al., (1990) Nucleic Acids Res 8:4057), andthe lambda-derived P_(L) promoter and N-gene ribosome binding site(Shimatake et al., (1981) Nature 292:128). However, any availablepromoter system compatible with procaryotes can be used.

The expression systems useful in the eukaryotic systems of the inventioncomprise promoters derived from appropriate eukaryotic genes. A class ofpromoters useful in yeast, for example, include promoters for synthesisof glycolytic enzymes, including alcohol dehydrogenase promoters,glyceraldehyde-3-phosphate dehydrogenase promoter (Holland & Holland,(1980) J Biol Chem 25:2596), alpha-factor promoter (Bitter et al.,(1984) Proc Natl Acad Sci 81:5330), the gal promoter (Johnston & David,(1984) Mol Cell Biol 4:1440) those for 3-phosphoglycerate kinase(Hitzeman et al., (1980) J. Biol Chem 256:1385) or the Leu2 geneobtained from YEp13 (Broach, J., et al., (1978) Gene 8:121).

Suitable mammalian promoters include the early and late promoters fromSV40 (Fiers et al., (1978) Nature 273:113) or other viral promoters suchas those derived from polyoma, adenovirus II, bovine papilloma virus oravian sarcoma viruses. Suitable viral and mammalian enhancers are citedabove. In the event plant cells are used as an expression system, thenopaline synthesis promoter is appropriate (Depicker, A., et al., (1982)J Mol Appl Gen 1:56).

The expression systems are included on replication vectors or are causedto integrate into the chromosome of a recombinant host. For systemswherein the vectors include a replication system, these may be low orhigh copy number, usually having copy numbers of fewer than about 1000,although in certain situations, runaway vectors may be employed. Whetherprovided on a vector intended for integration or in a replicationsystem, the sequence encoding a polypeptide of the invention may beligated in tandem with an amplifying gene such as dihydrofolatereductase, metallothioneins, thymidine kinase, or the like. Inprocaryotic systems, both the amplifying gene and the target gene can beunder the regulation of the same transcriptional and translationalregulatory regions.

Usually, the vector will include a marker which allows for selection ofhost cells containing the expression system; the nature of these markersdepends on the host and is understood in the art. In addition torequired regulators such as promoters, additional sequences such asenhancers can also be employed to enhance the level of transcription. Ifthe polypeptide is to be secreted, an upstream sequence encoding signalpeptides such as those described in U.S. Pat. Nos. 4,336,336; 4,338,397;and 4,546,082 may be employed. The signal sequence is enzymaticallycleaved as the polypeptide product is secreted.

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., (1972) Proc Natl AcadSci USA 69:2110; or the RbCl method described in Maniatis et al.,Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Press,p. 254 is used for procaryotes or other cells which contain substantialcell wall barriers. Infection with Agrobacterium tumefaciens (Shaw, C.H., (1938) et al., Gene 23:315) is used for certain plant cells. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, (1978) Virology 52:2:546is preferred. Transformations into yeast are carried out, for example,according to the method of Van Solingen, P., et al., (1977) J Bacter130:946; and Hsiao, C.L., et al., (1979) Proc Natl Acad Sci USA 76:3829.

In general, after construction of a suitable expression system, thesystem is transfected into the appropriate host and successfultransformants are selected by markers contained on the expressionvectors. Successfully transformed colonies are then cultured in order toproduce the desired polypeptide. It is sometimes preferred that apromoter which can be controlled by regulating conditions in theenvironment be used so that the cells can be grown under conditionswhere the gene encoding the desired polypeptide of the invention is notexpressed, and then production of the polypeptide induced by appropriatemanipulation of conditions. For example, if the trp promoter is used inE. coli, the cells are grown in the presence of tryptophan andexpression is then induced by diminution of tryptophan concentration orby addition of a tryptophan analogue such as indolylacetic acid. If thegene is under control of the PL promoter, the cells are grown atrelatively low temperature, such as at about 35° C., to a suitable celldensity, and the temperature is then elevated to activate this promoter.If produced in bacterial hosts as a mature intracellular polypeptide,the N-terminal methionine may or may not be cleaved. In mammaliansystems, for example, the use of the metallothionein promoter permitsinduction by addition of heavy metals or glucocorticoids. This protocolis preferred to prevent premature accumulation of the polypeptide whichmight be harmful to the growth of the cell.

The polypeptide can be produced intracellularly, or in secreted form byconstruction of vectors in which the peptide is preceded by a signalpeptide workable in the appropriate host.

The polypeptide is recovered from the medium or from the cells usingsuitable techniques generally known in the art, and purified by, forexample, ion exchange chromatography, ammonium sulfate precipitation,gel permeation chromatography, and so forth.

It will of course be understood, that antibodies to any of thepolypeptides disclosed herein could be generated, as described inconnection with the "normal" polypeptide (SEQ ID NO:1). Methodology andproducts can be developed using an antibody to a polypeptide for use indetecting the polypeptide with which the antibody binds. Methodology andproducts can be developed using an antibody to a polypeptide for use indetecting the polypeptide with which the antibody binds.

For example, an antibody can be linked to or conjugated with a reportersystem which is set up to indicate positively binding of the polypeptideto the antibody. Well known reporter systems include radioimmuno assays(RIAs) or immunoradiometric assays (IRMAs). Alternatively, anenzyme-linked immunosorbent assay (ELISA) would have in common with RIAsand IRMAs a relatively high degree of sensitivity, but would generallynot rely upon the use of radioisotopes. A visually detectable substancemay be produced or at least one detectable in a spectrophotometer. Anassay relying upon fluroescence of a substance bound by the enzyme beingassayed could be used. It will be appreciated that there are a number ofreporter systems which may be used, according to the present invention,to detect the presence of a particular polypeptide. With standardizedsample collection and treatment, polypeptide presence above a thresholdamount in blood serum could well be determined.

Such an antibody-linked reporter system could be used in a method fordetermining whether blood serum of a subject contains a deficient amountof the polypeptide. Given a normal threshold concentration of such apolypeptide in blood serum of a given type of subject, test kits couldthus be developed.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 33                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 36 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys Ile Lys         #                15                                                           - Pro Asn Thr Leu His Lys Lys Ala Ala Glu Th - #r Leu Met Val Leu Asp         #            30                                                               - Gln Asn Gln Pro                                                                     35                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 36 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is N-acetyl glycine."                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Xaa Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys Ile Lys         #                15                                                           - Pro Asn Thr Leu His Lys Lys Ala Ala Glu Th - #r Leu Met Val Leu Asp         #            30                                                               - Gln Asn Gln Pro                                                                     35                                                                    - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 36 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Ala Lys Ile Lys         #                15                                                           - Pro Asn Thr Leu His Lys Lys Ala Ala Glu Th - #r Leu Met Val Leu Asp         #            30                                                               - Gln Asn Gln Pro                                                                     35                                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 30 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys Ile Lys         #                15                                                           - Pro Asn Thr Leu His Lys Lys Ala Ala Glu Th - #r Leu Met Val                 #            30                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 25 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys Ile Lys         #                15                                                           - Pro Asn Thr Leu His Lys Lys Ala Ala                                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys Ile Lys         #                15                                                           - Pro Asn Thr Leu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys Ile             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Gly Ile Gly Lys Arg Thr Asn Glu His Thr Al - #a Asp Cys Lys                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Arg Thr Asn Glu His Thr Ala Asp Cys Lys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Leu His Lys Lys Ala Ala Glu Thr Leu Met Va - #l Leu Asp Gln Asn Gln         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Leu His Lys Lys Ala Ala Glu Thr Leu Met Va - #l Leu Asp Gln Asn             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Leu His Lys Lys Ala Ala Glu Thr Leu Met Va - #l Leu Asp Gln                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Leu His Lys Lys Ala Ala Glu Thr Leu Met Va - #l Leu Asp                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Le - #u His Lys Lys Ala Ala         #                15                                                           - Glu Thr Leu Met Val Leu Asp                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 30 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Arg Thr Asn Glu His Thr Ala Asp Cys Lys Il - #e Lys Pro Asn Thr Leu         #                15                                                           - His Lys Lys Ala Ala Glu Thr Leu Met Val Le - #u Asp Gln Asn                 #            30                                                               - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Arg Thr Asn Glu His Thr Ala Asp Cys Lys Il - #e                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 90 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - CGGAA AACGAACAAA TGAACATACG GCAGATTGTA AAATTAAACC GAACACC - #TTG            60                                                                            #           90     AGAC TTTAATGGTC                                            - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 75 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - CGGAA AACGAACAAA TGAACATACG GCAGATTGTA AAATTAAACC GAACACC - #TTG            60                                                                            #    75                                                                       - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - GGGATCGGAA AACGAACAAA TGAACATACG GCAGATTGTA AAATTAAACC GA - #ACACCTTG         60                                                                          - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #45                CAAA TGAACATACG GCAGATTGTA AAATT                           - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #  42              CAAA TGAACATACG GCAGATTGTA AA                              - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #           30     CGGC AGATTGTAAA                                            - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 108 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - GGGATCGGAA AACGAACAAA TGAACATACG GCAGATTGTA AAATTAAACC GA - #ACACCTTG         60                                                                          #               108AGAC TTTAATGGTC CTTGACCAAA ATGAACCA                        - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is N-acetyl arginine."                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/product= "Other"R INFORMATION:                                              #"The Xaa at position 10 is lysinamide."                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Xaa Thr Asn Glu His Thr Ala Asp Cys Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is N-acetyl arginine."                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/product= "Other"R INFORMATION:                                              #"The Xaa at position 10 is lysinamide."                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Xaa Thr Asn Glu His Thr Ala Glu Cys Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is N-acetyl arginine."                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/product= "Other"R INFORMATION:                                              #"The Xaa at position 10 is lysinamide."                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Xaa Thr Gln Glu His Thr Ala Glu Cys Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is N-acetyl arginine."                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/product= "Other"R INFORMATION:                                              #"The Xaa at position 10 is lysinamide."                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Xaa Thr Gln Glu His Thr Ala Asp Cys Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Arg Thr Asn Glu His Thr Ala Glu Cys Lys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Arg Thr Gln Glu His Thr Ala Glu Cys Lys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Arg Thr Gln Glu His Thr Ala Asp Cys Lys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #          30      AGAATGTAAA                                                 - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #          30      AGAATGTAAA                                                 - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #         30       TGTAAA                                                     __________________________________________________________________________

What is claimed is:
 1. A synthetic polypeptide having the amino acidsequence identified as SEQ ID NO:1 wherein at least one amino acid hasbeen deleted therefrom provided that amino acids 5 to 14 of SEQ ID NO:1are not deleted, or a substituted variant thereof in which (i) anon-polar, aliphatic neutral amino acid has been substituted for anothernon-polar, aliphatic neutral amino acid, (ii) a polar aliphatic neutralamino acid has been substituted for another polar aliphatic neutralamino acid, (iii) a charged acidic amino acid has been substituted foranother charged acidic amino acid, (iv) a charged basic amino acid hasbeen substituted for another charged basic amino acid, (v) cysteine hasbeen substituted by alanine, or (vi) a combination of substitutions(i)-(v) has been made, whereby said synthetic polypeptide has bonestimulatory activity in animals and which increases mineral content inbones of animals.
 2. A synthetic polypeptide of claim 1 wherein at least6 amino acids have been deleted from SEQ ID NO:1.
 3. A syntheticpolypeptide of claim 1 wherein at least 16 amino acids have been deletedfrom SEQ ID NO:1.
 4. A synthetic polypeptide of claim 1 wherein at least21 amino acids have been deleted from SEQ ID NO:1.
 5. A syntheticpolypeptide of claim 1 wherein 26 amino acids have been deleted from SEQID NO:1.
 6. A chimeric bone stimulating factor comprising a syntheticpolypeptide of claim
 1. 7. A method of increasing bone growth in amammal comprising administering to said mammal an amount of thesynthetic polypeptide according to claim 1 effective to increase bonegrowth in said mammal.